Glutamate and y-aminobutyric acid (GABA) are two major fast neurotransmitters (excitatory and inhibitory, respectively) in most regions of the central nervous system (CNS), including the hypothalamus. Together, they play a key role in the control of the activity and excitability of neurons and determine the synaptic excitation/inhibition balance in many neuronal circuits. Selective degeneration of glutamate-containing projecting neurons occurs in the human hippocampus and cerebral cortex during epilepsy, Alzheimer?s disease, ischemia, and cardiorespiratory arrest. Our previous experiments revealed that a chronic blockade of ionotropic glutamate receptors dramatically increased the expression of acetylcholine (ACh) neurotransmission in rat hypothalamic neuronal cultures. The increase in ACh activity following the decrease in glutamate excitation was associated with the up-regulation of ACh receptors. This demonstrates the important role of NMDA glutamate receptor blockade (but not non-NMDA receptor blockade or activity-dependent mechanisms) for the induction of cholinergic activity. Additionally, the data suggest that in the absence of glutamate excitation in hypothalamic cultures, Ach, another excitatory neurotransmitter, supports the excitation/inhibition balance. We therefore postulate that during a long-term decrease in the glutamate excitation in hypothalamic neuronal cultures, ACh, which normally exhibits a weak activity in the hypothalamus, begins to play the role of the major excitatory neurotransmitter and to support the excitation/inhibition balance. We also hypothesize that the increase in excitatory ACh transmission in the absence of glutamate excitation is a form of neuronal plasticity that regulates the activity and excitability of neurons during the glutamate/GABA imbalance. In our future research, we will address Ca2+ mechanisms of glutamate-dependent regulation of ACh transmission in the hypothalamus in vitro.